Comparison of ent-kaurene and ent-isokaurene synthesis in cell-free systems from etiolated shoots of normal and dwarf-5 maize seedlings

An active cell-free system, prepared from young etiolated shoots of normal Zea mays seedlings, was shown to biosynthesize the terpenoid hydrocarbons ent-kaur-16-ene, squalene and phytoene from mevalonic acid. The biosynthesis of ent-kaur-16-ene from mevalonic acid was compared using cell-free systems obtained from normal and dwarf-5 seedlings. ent-Kaur-16-ene was the predominant diterpene hydrocarbon synthesized by extracts from the normals; however, ent-kaur-15-ene was the major diterpene hydrocarbon synthesized by the dwarf-5 mutants. ent-Kaur-15-ene and ent-kaur-16-ene were also produced as minor products in the normal and dwarf-5 systems, respectively. The possible significance of the synthesis of the ‘wrong isomer’ (ent-kaur-15-ene) by the mutant is discussed.     

Synthesis of monoterpene hydrocarbons from [1-3H]linalyl pyrophosphate by carbocyclase from Citrus limonum

A partially purified enzyme preparation from the flavedo of Citrus limonum utilized [1-³H]linalyl pyrophosphate as a substrate for cyclic terpene hydrocarbon formation more efficiently than the pyrophosphates of nerol and geraniol. The products formed from all three substrates are α-pinene, β-pinene, limonene, and γ-terpinene. Neryl and geranyl pyrophosphate inhibit the formation of these products from linalyl pyrophosphate. No free linalyl pyrophosphate could be detected during the enzymatic formation of cyclic terpene hydrocarbons from geranyl pyrophosphate. Mn²⁺ catalyzes the nonenzymatic solvolysis of linalyl pyrophosphate, forming myrcene and ocymenes and no bicyclic hydrocarbons. Linalyl pyrophosphate is a sterically plausible precursor of cyclic hydrocarbons, but the present data support only its role as an alternative substrate and not as an obligatory free intermediate in terpene biosynthesis.     

Biosynthesis of rice phytoalexins: identification of putative diterpene hydrocarbon precursors.

A procedure for the preparation of a cell-free enzyme solution from rice leaves capable of catalyzing the biosynthesis of diterpene hydrocarbons from geranylgeranyl pyrophosphate or copalyl pyrophosphate as added substrates has been developed. The rates of synthesis of a group of "pimaradiene-like" diterpene hydrocarbons are about 75-fold higher with geranylgeranyl pyrophosphate as substrate and about 8-fold higher with copalyl pyrophosphate as substrate in comparison with extracts from untreated control leaves. The maximum rate of diterpene hydrocarbon biosynthesis is seen in extracts prepared at 40 h after uv irradiation. Five diterpene hydrocarbons (compounds A-E) were present in the hydrocarbon fraction biosynthesized from [3H]geranylgeranyl pyrophosphate in large-scale incubation mixtures prepared from uv-treated rice leaves. Three of these diterpenes were identified as ent-kaur-16-ene (B), ent-sandaracopimara-8(14), 15-diene (D), and 9 beta H-pimara-7,15-diene (E) from GC retention times and GC-MS spectral characteristics in comparison with those of authentic reference compounds. Compound C has spectral characteristics analogous to those of a pimaradiene, but a specific structural assignment from the data available was not possible. Similar incubations with [3H]copalyl pyrophosphate as the substrate and enzyme prepared from uv-treated rice leaves produced ent-kaurene (B), ent-sandaracopimara-8(14),15-diene (D), and compound C, but not 9 beta H-pimara-7,15-diene (E). These results are consistent with a proposed biosynthetic scheme in which 9 beta H-pimara-7,15-diene serves as a precursor of the momilactone family, and ent-sandaracopimara-8(14),15-diene serves as a precursor of the oryzalexin family of rice phytoalexins. ent-Kaurene was the only diterpene detected in incubation mixtures containing enzyme extract from untreated rice leaves and [3H]copalyl pyrophosphate as the substrate. It is suggested that kaurene biosynthesis in rice leaves is probably associated with gibberellin biosynthesis and the regulation of vegetative growth rather than stress metabolism. The diterpene cyclization enzymes in extracts of uv-treated rice leaves show only a relatively modest inhibition by the plant growth retardants AMO-1618 and Phosfon D. No evidence was obtained for the subcellular localization of these cyclization enzymes in organellar preparations; it is tentatively concluded that the enzymes are present predominantly in the extraorganellar cytoplasm of rice leaves.     

Biosynthesis of labdenediol and sclareol in cell-free extracts from trichomes of Nicotiana glutinosa

Biosynthesis of the diterpenes labdenediol and sclareol from all trans-geranylgeranyl pyrophosphate was observed in cell-free extracts prepared from leaf midvein epidermal peels of Nicotiana glutinosa 24A. The bioactivities were shown to be exclusively localized in trichomes, to be conferred by a soluble enzyme(s), and to resemble other plant terpene cyclase activities in basic characteristics. Chromatographic methods for protein purification including gel filtration, anion exchange, hydroxyapatite chromatography, and free-flow isoelectric focusing. Thermal inactivation and end-product inhibition experiments did not afford separation of the biosynthetic activities. Results indicate that these labdane diterpenes are direct products of cyclization reactions catalysed by a soluble cyclase(s). To the best of our knowledge, this report represents the first case for direct biosynthesis of diterpene di-alcohols by cyclization of the acyclic precursor. A unified scheme for formation of labdane mono- and di-alcohols in Nicotiana species is presented.Abbreviations DEAE diethylaminoethyl - GGPP geranyl-geranyl pyrophosphate     

Three ent-kaurene diterpenes from Vellozia caput-ardeae

Three new ent-kaurene diterpenes have been isolated from the roots and stem of Vellozia caput-ardeae. Their structures were elucidated by spectroscopic methods as ent-9β-hydroxy kaur-16-ene, ent-11α-hydroxy kaur-16-ene and ent-9β,11α-dihydroxy kaur-16-ene.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : Changes in Enzyme Levels Induced by Fungal Infection and Intracellular Localization of the Pathway

Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.     

Fusicocca-3(16),10(14)-diene, and beta- and delta-araneosenes, new fusicoccin biosynthesis-related diterpene hydrocarbons from Phomopsis amygdali

Further isolation and examination of fusicoccane hydrocarbons biosynthetically related to fusicoccin from Phomopsis amygdali allowed us to identify new fungal diterpene hydrocarbons of fusicoccadiene and araneosene. These were assigned as (+)-fusicocca-3(16),10(14)-diene, and (+)-beta- and (+)-delta-araneosenes. These findings led to the experimental clarification of the structures of the biosynthetic hydrocarbon intermediates presumed earlier.     

Characterization of undecaprenyl pyrophosphate synthetase from Lactobacillus plantarum

A soluble long-chain polyprenyl pyrophosphate synthetase has been isolated from Lactobacillus plantarum and partially purified by DEAE-cellulose chromotography in 1% Triton X-100. This enzyme catalyzes the synthesis of polyprenyl pyrophosphate from farnesyl pyrophosphate and Δ³-isopentenyl pyrophosphate. The enzyme displays a requirement for farnesyl pyrophosphate and Triton X-series detergents. Treatment of polyprenyl pyrophosphate with C₅₅-isoprenyl pyrophosphate phosphatase (Micrococcus lysodeikticus) yielded polyprenyl monophosphate. Subsequent treatment of this product with a crude phosphatase from baker's yeast resulted in the formation of free polyprenol, which was characterized by thin layer chromatography and exhibited R_fs which corresponded to those of authentic undecaprenol isolated from Lactobacillus plantarum. Reverse phase cochromatography of the enzymically produced polyprenol and authentic undecaprenol indicated that the major enzymic products were undecaprenol and probably a longer chain polyprenol.     

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.     

Biochemical synthesis of uniformly 13C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins in planta

Phytocassanes and momilactones are the major diterpenoid phytoalexins inductively produced in rice as bioactive substances. Regardless of extensive studies on the biosynthetic pathways of these phytoalexins, bioconversion of diterpene hydrocarbons is not shown in planta. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly ¹³C-labeled ent-cassadiene and syn-pimaradiene were enzymatically synthesized with structural verification by GC–MS and ¹³C-NMR. Application of the ¹³C-labeled substrates on rice leaves led to the detection of ¹³C-labeled metabolites using LC-MS/MS. Further application of this method in the moss Hypnum plumaeforme and the nearest out-group of Oryza species Leersia perrieri, respectively, resulted in successful bioconversion of these labeled substrates into phytoalexins in these plants. These results demonstrate that genuine biosynthetic pathways from these diterpene hydrocarbons to the end product phytoalexins occur in these plants and that enzymatically synthesized [U-¹³C₂₀] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds.     

Metabolism of kaurenoids by Gibberella fujikuroi in the presence of the plant growth retardant,N,N,N-trimethyl-1-methyl-(2′,6′,6′-trimethylcyclohex-2′-en-1′-yl)prop-2-enylammonium iodide

The plant growth retardant, N,N,N-trimethyl-1-methyl-(2′,6′,6′-trimethylcyclohex-2′-en-1′-yl)prop-2-enylammonium iodide, is shown to block gibberellin biosynthesis in Gibberella fujikuroi between mevalonate and ent-kaur-16-ene, probably by inhibiting ent-kaur-16-ene synthetase A-activity. In the presence of the plant growth retardant, cultures of the fungus incorporate (26.5%) added ent-[¹⁴C]-kaur-16-ene into gibberellin A₃. Under the same conditions kaur-16-ene, 13β-kaur-16-ene, and ent-kaur-15-ene are not metabolised to gibberellin analogues.     

Biosynthetic study of conidiation-inducing factor conidiogenone: heterologous production and cyclization mechanism of a key bifunctional diterpene synthase

Conidiogenone, a diterpene with a unique structure, is known to induce the conidiation of Penicillium cyclopium. The biosynthetic pathway of (?)-conidiogenone has been fully elucidated by the heterologous expression of biosynthetic genes in Aspergillus oryzae and by in vitro enzyme assay with ¹³C-labeled substrates. After construction of deoxyconidiogenol by the action of bifunctional terpene synthase, one cytochrome P450 catalyzes two rounds of oxidation to furnish conidiogenone. Notably, similar biosynthetic genes are conserved among more than 10 Penicillium sp., suggesting that conidiogenone is a common conidiation inducer in this genus. The cyclization mechanism catalyzed by terpene synthase, which involves successive 1,2-alkyl shifts, was fully elucidated using ¹³C-labeled geranylgeranyl pyrophosphate (GGPP) as substrate. During the structural analysis of deoxyconidiogenol, we observed broadening of some of the ¹³C signals measured at room temperature, which has not been observed with other structurally related compounds. Careful examination using techniques including ¹³C NMR studies at ?80 °C, conformational analysis and prediction of the ¹³C chemical shifts using density functional theory gave insights into this intriguing phenomenon.     

Substrate specificity of α‐humulene synthase from Zingiber zerumbet Smith and determination of kinetic constants by a spectrophotometric assay

Terpene synthases are the key enzymes in terpene biosynthesis that provide a structurally complex and highly diverse product spectrum. A suitable and reliable analytical assay is indispensable to measure terpene synthase activity accurately and precisely. In this study, a malachite green assay (MG) was adapted to rapidly assay terpene synthase activity and was validated in comparison to an already established gas chromatography assay. A linear correlation between both assays was observed. Kinetic properties for the previously described sesquiterpene synthase α‐humulene synthase (HUM) from Zingiber zerumbet Smith were investigated for the bioconversion of the monoterpene precursors geranyl pyrophosphate (2E‐GPP) and neryl pyrophosphate (2Z‐NPP) as well as for the sesquiterpene precursor farnesyl pyrophosphate (2E,6E‐FPP). Also, gas chromatography mass spectrometry (GS‐MS) was carried out to identify the products of the bioconversion of (2E)‐GPP and (2Z)‐NPP.     

The metabolism of a series of polycyclic hydrocarbons by mouse skin maintained in short-term organ culture

Investigations on the metabolism of ³H-labelled chrysene, benz[a]anthracene, 7-methylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, dibenz[a,c]anthracene and dibenz[a,h]anthracene by mouse skin maintained in short-term organ culture were carried out. Estimations of the distribution of the metabolites of each hydrocarbon present after 24 h showed that there were wide variations both in the rates at which the hydrocarbons were metabolised and in the amounts of metabolites covalently bound to skin macromolecules. All the hydrocarbons were metabolised to dihydrodiols, which were identified by comparison on high pressure liquid chromatography (HPLC) with the authentic compounds, and these were the same diols as those that were formed in previous experiments with rat-liver microsomal fractions. However, free dihydrodiols represented only relatively small proportions of the total amounts of metabolites formed. All the hydrocarbons yielded dihydrodiols of the type that could give rise to bay-region diol-epoxides, when further metabolised, some of which are thought to be involved in hydrocarbon carcinogenesis.     

Identification and characterization of the pseudopterosin diterpene cyclase, elisabethatriene synthase, from the marine gorgonian, Pseudopterogorgia elisabethae

The pseudopterosins are diterpene glycosides isolated from the marine gorgonian, Pseudopterogorgia elisabethae, which exhibit anti-inflammatory and analgesic activity greater than the industry standard, indomethacin. Previously, we isolated the pseudopterosin diterpene cyclase product, elisabethatriene, using a radioactivity-guided isolation. Identification of this metabolite, and the conversion of labeled geranylgeranyl diphosphate to elisabethatriene, provided us with an assay to guide the isolation of the enzyme responsible for this cyclization. The soluble protein preparation from P. elisabethae has been partially purified (approximately 15,000-fold) using a combination of low-resolution anion-exchange, low-resolution hydrophobic interaction, high-resolution hydroxyapatite, and high-resolution anion-exchange chromatography. The diterpene cyclase was identified by comparing the molecular weight from gel permeation chromatography (approximately 47,000Da) with those of protein bands from purified fractions using SDS-PAGE gel electrophoresis. Kinetic analysis and evaluation of amino acid inhibition studies indicated that the enzyme displays similar characteristics to other terpenoid cyclases isolated from terrestrial sources. This report represents the first purification and characterization of a terpene biosynthetic enzyme from a marine invertebrate.     

Glucosylation of Steviol and Steviol-Glucosides in Extracts from Stevia rebaudiana Bertoni

To evaluate and characterize stevioside biosynthetic pathway in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19-oic acid) and steviol-glucosides (steviol-13-O-glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glucose donor, were prepared from the soluble extracts of S. rebaudiana leaves. Enzyme fraction I, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-O-methylsteviol, but not to iso-steviol and 13-O-methylsteviol, indicating that 13-hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-O-glucopyranoside. Enzyme fraction II, eluted from the DEAE-Toyopearl column with 0.15 molar KCI, catalyzed the glucose transfer from UDP-glucose to steviol-13-O-glucopyranoside, steviolbioside and stevioside, but not to rubusoside (13, 19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-O-glucopyranoside, steviolbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indicate that in the steviol-glucoside biosynthetic pathway, steviol-13-O-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside producing rebaudioside A.     

Spirocyclopropane-type sesquiterpene hydrocarbons from Schinus terebinthifolius Raddi

The essential oil of Schinus terebinthifolius fruits was reinvestigated using GC and GC–MS techniques. Apart from several known compounds three sesquiterpene hydrocarbons with a carbon skeleton exhibiting the rare spiro(cyclopropane) moiety could be isolated by a combination of column chromatography and GLC. Structure assignments were carried out by NMR spectroscopy. These natural products are 9-spiro(cyclopropa)-4,4,8-trimethyl-2-methylenbicyclo[4.3.0]non-1(6)-ene (terebanene), 9-spiro(cyclopropa)-2,4,4,8-tetramethylbicyclo[4.3.0]nona-1,5-diene (teredenene), and (6R1,8R1)-9-spiro(cyclopropa)-2,4,4,8-tetramethylbicyclo[4.3.0]non-1-ene (terebinthene).     

Within-plant distribution and emission of sesquiterpenes from Copaifera officinalis

Copaifera officinalis, the diesel tree, is known for massive production of oleoresin, mainly composed of sesquiterpene hydrocarbons. In this study, composition of these sesquiterpenes and their concentrations in leaves, stems and roots of C. officinalis at two developmental stages, including the three-week old (TW) seedlings and two-year old (TY) trees, were determined. The leaves of TW seedlings and TY trees contained similar number of sesquiterpenes, which also had comparable concentrations. The stems of TW seedlings had higher concentrations of sesquiterpenes than those of TY trees. In contrast, the number of sesquiterpene species and their concentrations in the roots of TW seedlings were much lower than those in the roots of TY trees. Cluster analysis of sesquiterpenes estimated that there are at least four terpene synthase genes involved in the production of sesquiterpenes in C. officinalis. Because sesquiterpenes are highly volatile, emissions of sesquiterpenes from healthy and wounded TW seedlings were examined using headspace analysis. Whereas very low emission of sesquiterpenes was detected from undamaged plants, the physically injured seedlings emitted a large number of sesquiterpenes, the quality and the relative quantity of which were similar to those in leaves determined using organic extraction. The implications of our findings to the biosynthetic pathways leading to the production of sesquiterpenes as well as their biological roles in C. officinalis are discussed.     

Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis

A plastid vesicle preparation isolated from exocarpium of young Citrofortunella mitis (calamondin) fruits was able to synthesise monoterpene hydrocarbons when incubated with isopentenyl pyrophosphate. The electron-microscope comparison between this organelle fraction and the various plastid classes present in the peel tissues has shown the structural identity between these plastid vesicles and the leucoplasts of the epithelial cells lining the secretory pockets. The monoterpene biosynthesis required the presence of dimethylallyl pyrophosphate, Mn²⁺ or Mg²⁺ and was increased by addition of 2-mercaptoethanol. Evidence is provided that the leucoplast vesicles act as a complete system in which occur all the successive steps involved in monoterpene hydrocarbon elaboration from isopentenyl pyrophosphate.